Alexa fluor 488 conjugated anti mouse immunoglobulin g

ARPE-19 cells were plated onto chamber slides, and then cells were fixed with 4% paraformaldehyde for 20 min and blocked with 5% skim milk for 1 h at room temperature. Cells were then incubated with the rabbit-anti-FTMT primary antibody (1:500) or mouse anti-pan cadherin antibody (1:200) (ab6528-Abcam, Cambridge, U.K.) overnight at 4 °C. After washing three times with TBST, cells were incubated with Alexa Fluor 568-conjugated anti-rabbit immunoglobulin G (1:1000; ThermoFisher) to detect FTMT or with Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (1:1000; ThermoFisher) for 1 h at room temperature; then, cells were counterstained with DAPI to allow for the identification of nuclei and cover-slipped using a fluorescent-mounting agent. Stained cells were examined using a confocal microscope (Leica SP8 Lightning, Leica, Wetzlar, Germany).

Cells were seeded onto a 18 mm × 18 mm glass coverslip (Matsunami) placed in a 6-well plate and grown overnight. Cells were then transfected with plasmid as specified in figure legends. Afterward, cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature (55 (link)). After blocking with 5% FBS in PBS for 1 h, cells were incubated with primary antibodies (anti-Tom20 monoclonal antibody from Cell Signaling Technology, catalog no.: 42406, anticalnexin monoclonal antibody from Cell Signaling Technology, catalog no.: 2679, or anti-FLAG monoclonal antibody from Sigma, F3165) for 1 h. After washing with 1% FBS in PBS three times, specimens were incubated with Alexa Fluor 488–conjugated antimouse immunoglobulin G (Invitrogen; A11029) and Alexa Fluor 568–conjugated anti-rabbit immunoglobulin G (Invitrogen; A11036) (1:800 dilution) in 2% FBS in PBS for 45 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Specimens were washed with PBS for three times and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Immunofluorescence confocal images were acquired using a Zeiss LSM800 with Airyscan equipped with a Plan-Apochromat 63×/1.40 Oil DIC M27 objective (Carl Zeiss). Images were processed with a Zen software (Carl Zeiss) and Fiji software (Fiji).

Cultured human monocytes were fixed with 4% formaldehyde in PBS for 20 min at RT. The cells were washed three times with PBS, permeabilized with 0.3% Triton X-100 for 30 min, and blocked in 10% goat serum in PBS for 2 h at RT, followed by incubation with CXCR3 antibody (Santa Cruz, Cat# sc-137140, Dallas, TX, United States) at 1:200 dilution. The cells were washed with PBS and incubated with Alexa Fluor 488–conjugated anti-mouse immunoglobulin G (Invitrogen) for 1 h at RT. After the final wash with PBS, cells were mounted using the mounting medium (Prolong Gold Antifade Reagent; Invitrogen) on the slides. Fluorescent images were acquired at RT on a Zeiss Observer, using a Z1 inverted microscope with a 63×/1.4 oil-immersion objective. Images were processed with the AxioVs 40 Version 4.8.0.0 software (Zeiss). Photographs were acquired with an AxioCam MRm digital camera and were analyzed using the ImageJ software.